AlphaLISA SureFire Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points

Phospho-AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.

Induction of STAT6 phosphorylation in PBMCs stimulated with various cytokines

PBMCs were isolated from healthy donors using Ficoll® Plaque Plus. Cells were seeded in a 96-well plate (400,000 cells/well) and starved for 2 hours in serum-free DMEM. Cells were then treated with the indicated cytokines for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) levels were evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, IL-4 was the main activator of STAT6 phosphorylation.

IL-4 induces STAT6 phosphorylation in a dose-dependent manner

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (20,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were treated with the indicated concentration of IL-4 for 20 minutes.

After treatment, cells were lysed in 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.

As expected, IL-4 triggered a dose-dependent increase in the levels of Phospho STAT6 (Tyr641) while Total STAT6 levels remained unchanged.

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1 % BSA and treated with increasing concentrations of IL-4 for 20 minutes.

After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 16,000 cells/datapoint) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, IL-4 triggered a dose-dependent increase in the levels of STAT6 Phospho Tyr641 while Total STAT6 levels remained unchanged.

HeLa cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of IL-4 for 20 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Total and Phospho (Tyr641) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, IL-4 triggered a dose-dependent increase in the levels of Phospho STAT6 (Tyr641) while Total STAT6 levels remained unchanged.